Comparison of binding of mixed ribose-deoxyribose analogues of CUCU to a ribozyme and to GGAGAA by equilibrium dialysis: evidence for ribozyme specific interactions with 2' OH groups

Dissociation constants at 15 degrees C were measured by equilibrium dialysis for the binding of rCrUrCrU, dCrUrCrU, rCdUrCrU, rCrUdCrU, and rCrUrCdU to the L-21 ScaI form of the self-splicing group I LSU intron from Tetrahymena thermophila. Substitution of deoxyribose for ribose in each of the middle two positions makes the free energy change for binding 1-2 kcal/mol less favorable, compared to about 0.3 kcal/mol less favorable for each of the terminal positions. Dissociation constants for binding of the same oligomers to rGGAGAA were measured by optical melting methods. Substitution of a single deoxyribose for ribose makes the free energy change for binding less favorable by 0.4-0.9 kcal/mol for this simple duplex formation. Comparison of the effects for binding to ribozyme and to rGGAGAA indicate that ribozyme-specific tertiary interactions dependent on the middle two 2' OH groups of rCrUrCrU add about 2 kcal/mol of favorable free energy for binding to L-21 ScaI. Comparisons are made with results from gel retardation studies [Pyle, A. M., McSwiggen, J. A., & Cech, T. R. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 8187-8191; Pyle, A. M., & Cech, T. R. (1991) Nature (London) 350, 628-631].     

Influence of acclimation and determination temperature on the oxygen consumption of the brain in two species of anurans

1. 1. The effects of sudden changes by increasing or decreasing the measurement temperature on the oxygen consumption of the brains of Bufo arenarum and Leptodactylus ocellatus were determined.

2. 2. The experiments were carried at in vitro at temperatures which range from 4 to 37°C. The brain was oxygenated and stabilized for 20 min at each of the temperatures to which it was subjected before oxygen consumption measurements were made.

3. 3. A theoretical curve representing the variation of oxygen consumption with temperature was calculated according to the following exponential relationship; for Leptodactylus ocellatus y = 0.408 × 1.07^x and for Bufo arenarum y = 0.389 × 1.065^x.

4. 4. These results were compared with the brain oxygen consumption of animals acclimated to different temperatures, whose oxygen consumption was measured at a fixed temperature. Only Leptodactylus ocellatus had a significantly lower oxygen consumption in a high range of temperatures, indicating thermal compensation, probably to save metabolic reserves.

5. 5. No deterioration of the brain tissue was observed, as several passages from high to low temperatures in the range of 20°–30°C, showed a reversible oxygen consumption in acclimated and non-acclimated Bufo arenarum and Leptodactylus ocellatus.

Study of the interaction of polyols with polymers containing n-substituted [(4-boronophenyl)methyl]-ammonio groups

Polymers, based on dextran and cellulose, having 2-{[(4-boronophenyl)-methyl]-ethylammonio}ethyl and -diethylammonio～ethyl groups were prepared. It was shown that these polymers could be employed for absorption of cis-diol compounds. The polymers were found to be highly specific towards polyols, carbohydrates, nucleosides, and nucleotides over a wide range of pH. The polymer based on DEAE-Sephadex A-25 was used in separating nucleosides, and in fractionating mononucleotides and carbohydrates. The chromatographic behavior of carbohydrates is defined by their structure and conformation, which are also responsible for different stabilities of the boronic complexes generated.     

Optimising Immunogenicity with Viral Vectors: Mixing MVA and HAdV-5 Expressing the Mycobacterial Antigen Ag85A in a Single Injection

The Bacillus Calmette - Guerin (BCG) vaccine provides a critical but limited defense against Mycobacterium tuberculosis (M.tb). More than 60 years after the widespread introduction of BCG, there is an urgent need for a better vaccine. A large body of pre-clinical research continues to support ongoing clinical trials to assess whether viral vectors expressing M.tb antigens that are shared by BCG and M.tb, can be used alongside BCG to enhance protection. A major focus involves using multiple unique viral vectors to limit anti-vector immunity and thereby enhance responses to the insert antigen delivered. The successful introduction of viral vector vaccines to target M.tb and other pathogens will be reliant on reducing the costs when using multiple vectors and inhibiting the development of unwanted anti-vector responses that interfere with the response to insert antigen. This study examines methods to reduce the logistical costs of vaccination by mixing different viral vectors that share the same insert antigen in one vaccine; and whether combining different viral vectors reduces anti-vector immunity to improve immunogenicity to the insert antigen. Here we show that a homologous prime-boost regimen with a mixture of MVA (Modified Vaccinia virus Ankara) and Ad5 (human adenovirus type 5) vectors both expressing Ag85A in a single vaccine preparation is able to reduce anti-vector immunity, compared with a homologous prime-boost regimen with either vector alone. However, the level of immunogenicity induced by the homologous mixture remained comparable to that induced with single viral vectors and was less immunogenic than a heterologous Ad5 prime-MVA-boost regimen. These findings advance the understanding of how anti-vector immunity maybe reduced in viral vector vaccination regimens. Furthermore, an insight is provided to the impact on vaccine immunogenicity from altering vaccination methods to reduce the logistical demands of using separate vaccine preparations in the field.     

Effect of antioxidants on the genetic stability of cryopreserved mint shoot tips by encapsulation–dehydration

The effect of antioxidants applied in one step of a cryopreservation protocol by encapsulation–dehydration on recovery and genetic stability of mint shoot tips has been studied. Glutathione (0.16 or 0.24 mM), ascorbic acid (0.28 or 0.43 mM) and α-tocopherol (vitamin E) were added to the preculture medium (0.3 M sucrose). DNA was extracted from three different types of samples: leaves from shoots, callus at the base of shoots and callus. RAPD and AFLP markers were used to assess the genetic stability. The use of antioxidants did not improve recovery after cryopreservation. One of the genotypes, ‘MEN 198’, showed higher percentage of stable samples than the other one, ‘MEN 186’ (56 vs. 37?%; considering all treatments and types of explant). The use of vitamin E improved the percentage of stable samples with respect to control treatment (no antioxidant) in ‘MEN 186’. No differences in the percentages of stable samples were observed among cryopreserved and non-cryopreserved (treated similarly without immersion in liquid nitrogen) plant material. Recovered shoots of both genotypes showed higher stability (76–80?% stable samples) than callus samples (14–22?%).     

Plasmid transformation of Ruminococcus albus by means of high-voltage electroporation

To apply recombinant DNA techniques to the genetic manipulation of cellulolytic ruminal bacteria, a plasmid vector transformation system must be available. The objective of this work was to develop a system for plasmid transformation of Ruminococcus albus. Using high voltage electrotransformation, pSC22 and pCK17 plasmid vectors, derived from lactic acid bacteria plasmids and replicating via single-stranded DNA intermediate, were successfully introduced into three freshly isolated R. albus strains and into R. albus type strain ATCC 27210. The optimization of the electrotransformation condition raised the electroporation efficiency up to 3 x 10(5) transformants per microgram of pSC22 plasmid.